Method of cell culture using a dual circuit, woven artificial capillary bundle

ABSTRACT

A method for cell culture using an artificial capillary cell culture device having two separate perfusion circuits, and with the ultrafiltration fibers of the two circuits being woven into a single bundle. The present device provides a maxtrix for high density growth of cultured cells in a continuous perfusion system. The device is particularly well adapted for the use of pressure differences between perfusion circuits,, such as in the simulation of lymphatic drainage.

This is a divisional of application Ser. No. 850,810, filed Nov. 11,1977.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention is concerned with a method for using an artificialcapillary cell culture device. More particularly, the present inventionrelates to a method of use of an artificial capillary cell culturedevice having improved features which include the incorporation into asingle bundle of ultrafiltration fibers connected to two separateperfusion circuits and the interweaving of fibers to maintain intimatecontact. The artificial capillary device of the present inventive methodprovides a matrix for high density growth of cultured cells in acontinuous perfusion system.

Previous artificial capillary cell culture devices have included thedevice described in U.S. Pat. No. 3,883,393 to Knazek et al., in whichthere is provided a bundle of unwoven hollow fibers connected to asingle perfusion circuit. In such devices, in which the fibers are laidout parallel to one another during construction of the bundle, there arethree main problems which are inherent. One problem is that the fibersmay splay apart from one another when the bundle is sealed in the shell,increasing the possibility that cells between fibers may be anoxic, thuspreventing the formation of a continuous tissue-like cell mass on thefiber bundle. A second main problem resides in the fact that the fibersmay dry and contract following humidified ethylene oxide sterilizationof the device, one result being that breakage may occur more easily inindividual fibers. A third problem concerns distribution of nutrientswhich occurs simply by diffusion in the previous device.

By the present invention, there is provided an artificial capillary cellculture device which has overcome the difficulties described inconnection with previous cell culture devices such as that of U.S. Pat.No. 3,883,393. The cell culture device of the present invention hasfeatures which include the incorporation into a single woven bundle ofultrafiltration fibers, with a portion of the fibers being connected toone perfusion circuit and the remaining fibers being connected to asecond perfusion circuit. A difference in pressure between the twocircuits produce, connective currents of perfusate within theextracapillary space and improves nutrient distribution. Thus thepresent device is provided with two separate perfusion circuits whichfeed a single culture unit, but with the fibers associated with theseparate circuits being in intimate contact with one another in a wovenconfiguration. The present construction permits observation of masstransfer of chemical species from one perfusion circuit into anotherthrough the cell mass. In addition, the present device provides forinduction of fluid filtration between perfusion circuits to simulateprocesses such as lymphatic drainage of the cell mass.

The woven construction of the present fiber bundle results in a fiberbundle which is coherent and yet compliant, with the fibers from theseparate circuits being in intimate contact. In addition, while theweaving of the fiber bundle provides structural strength, at the sametime the bundle is sufficiently flexible to absorb possible contractionsor displacements caused by the imposition of fluid pressure differencesrequired to employ the device in processes such as inducing simulatedlymphatic drainage.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features of the present invention will be more fullyunderstood from the following description of the preferred embodiments,taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a perspective view of a cell culture unit of the presentinvention;

FIG. 2 is an exploded perspective view of the cell culture unit of FIG.1, showing various components thereof, prior to assembly and insertionof the woven capillary fiber bundle;

FIG. 3 is a front elevational view of the fiber bundle jig used to weavethe fibers; and

FIG. 4 is a perspective view of the fiber bundle jig of FIG. 3, showingthe fiber attachment points.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the embodiment of the invention as shown in FIGS. 1 and 2, a cellculture unit (CCU) 10 is provided, having a shell member 11 connected ateach end to a Y-connector endpiece 12 by means of connecting sleeves 13.A woven capillary fiber bundle 14 extends the length of the shell 11with portions of the bundle 14 extending through to the ends 15 of theendpieces 12. A pair of inoculation ports 16 of conventionalconstruction are provided to allow excess to the extracapillary spacethrough holes 17 having a diameter such as, for example, about 55 mil.

The shell member 11 is preferably of a clear, durable material such aspolycarbonate to provide visibility of the woven bundle 14 within theshell 11. The dimensions of the shell 11 may be, for example, a lengthof 5 cm with 1/4" I.D. and 3/8" O.D.

The endpieces 12 may be of a material such as standard Pyrex glassY-connectors having dimensions such as, for example, 3/16" I.D. and5/16" O.D., and cut down to yield a pair of arms of about 16 mm inlength. Thus there are provided a pair of arms 18, 19 at one end, and apair of arms 20, 21 at the opposite end of the unit 10. The baseportions 22, 23 of the endpieces 12 may be about 13 mm in length.

The connecting sleeves 13 may be formed of a material such as Silastictubing, a Dow-Corning silicone rubber medical grade tubing, of a lengthof about 17-20 mm with 1/4" I.D. and 3/8" O.D. These sleeves 13 areemployed to hold the endpieces 12 butted against the shell 11.

As described hereinafter, potting material 24 is employed to fill theendpieces 12 and intrude approximately 5 mm into the shell 11 at eachend. The potting material 24 may be any suitable composition employed inthe art. One composition which has been employed with good resultsincludes 60 wt.% General Electric Co. RTV (Room temperaturevulcanizing)--11 silicone rubber and 40 wt.% Dow-Corning 360 medicalfluid. This mixture is catalyzed with stannous octoate and aspiratedinto the endpieces. When the potting material 24 is trimmed away fromthe outer ends 15 of the endpieces 12, the capillary lumens 25 of thefibers making up the bundle 14 are revealed.

When the capillary fibers have been installed within the unit 10 asdescribed in detail hereinafter, one set of capillaries, such as about50 capillaries, is potted through arms 19 and 21 while a second set ofabout 50 capillaries is potted through arms 18 and 20. Thus oneperfusion circuit (not shown) may be employed to pump fluid from arm 19to arm 21 through one set of capillaries while a second perfusioncircuit (not shown) pumps fluid from arm 18 to arm 20.

In FIGS. 3 and 4 there is shown the fiber bundle jig 30 employed inproducing the woven fiber bundle 14. The jig 30 includes a pair of rods31 clamped in vertical position on any suitable base (not shown) andwith holes drilled in the upper ends of the rods 31 to accomodate astainless steel wire mandrel 32. The rods 31 may be formed, for example,of polycarbonate material having a diameter of about 1/2", while themandrel 32 has a diameter of about 1/16. A pair of spreader collars 33are installed on the mandrel 32 as shown in FIGS. 3 and 4. Thesespreader collars 33 may be formed, for example, of nested Silastictubing sections with 1/8" O.D. ×1/16" I.D. tubing on the mandrel 32,then a 2/10" O.D. ×1/10" I.D. tubing over the 1/16" tubing and finally a5/16" O.D. ×3/16" I.D. tubing over the 1/10" tubing. The distance "A"between spreader collars 33 may be about 4.8 cm, for example, with thedistance "B" between vertical rods 31 being about 13.2 cm. Externalslip-on collars 34 of a material such as 1/16" I.D. Silastic tubing, areslipped over the mandrel 32 on each end and snugged up adjacent theouter sides of the rods 31.

In the perspective view of FIG. 4, there are shown the four fiberattachment points 1,2,3,4 on the rods 31. Point 1 (left front) and point2 (left back) and correspondingly, point 3 (right front) and point 4(right back), are diametrically opposed sites on the upright rods 31where the mandrel 32 passes through.

The fibers employed in making up the bundle 14 may be any suitablehollow ultrafiltration fibers, such as those made by the AmiconCorporation of Lexington, Mass. Such semi-permeable fiber materials arefurther described in U.S. Pat. No. 3,883,393 which is incorporatedherein by reference.

In preparing the woven fiber bundle 14 of the present invention, aswatch of double-sided tape is applied to each of the four fiberattachment sites 1,2,3 and 4 on the uprights 31 of the bundling jig 30.The fibers are applied one at a time to the bundle, and the count offibers is kept on a hand tally counter or other suitable means. Afifteen-centimeter length of fiber is cut from a spool of fiber. One endis secured to the tape swatch at position 3. Next, the fiber is passedto position 2, and two turns are taken around the mandrel 32 in aclockwise sense (reference point--looking from the right upright alongthe mandrel 32 to the left upright) before the fiber is secured to thetape swatch at position 2. This process is repeated for a total of fivefibers. Then a fiber is attached to position 1 and passed to position 4,and two turns are wound counterclockwise around the mandrel 32 enroute.Repeat this tack until a total of ten fibers have been applied. Then,return to the 3-to-2 tack for another five fibers. After every fivefibers, switch tacks. Also, at each multiple of eleven fibers, thewinding sense is reversed. In addition, the winding sense for the 1-to-4tack is always opposite to that for the previous 3-to-2 tack, andvice-versa. Finally, after sixty fibers have been applied, the number ofturns is reduced to one. A total of one hundred fibers is bundled. It isadvantageous to apply fresh tape swatches to the attachment sites afterevery twenty fibers.

A tabulation of this bundling procedure follows:

                  TABLE 1                                                         ______________________________________                                        TACK                    NUMBER OF TURNS                                       (origin-to-             WRAPPED AROUND                                        destination)                                                                           FIBER NUMBERS  MANDREL                                               ______________________________________                                        3-to-2   1-5            2                                                     1-to-4    6-10          2                                                     3-to-2   11-15          2                                                     1-to-4   16-20          2                                                     apply fresh tape swatches                                                     3-to-2   21-25          2                                                     1-to-4   26-30          2                                                     3-to-2   31-35          2                                                     1-to-4   36-40          2                                                     apply fresh tape swatches                                                     3-to-2   41-45          2                                                     1-to-4   46-50          2                                                     3-to-2   51-55          2                                                     1-to-4   56-60          2                                                     apply fresh tape swatches                                                     3-to-2   61-65          1                                                     1-to-4   66-70          1                                                     3-to-2   71-75          1                                                     1-to-4   76-80          1                                                     apply fresh tape swatches                                                     3-to-2   81-85          1                                                     1-to-4   86-90          1                                                     3-to-2   91-95          1                                                     1-to-4    96-100        1                                                     ______________________________________                                    

A tabulation of the winding sense of the successive fibers is asfollows:

                  TABLE 2                                                         ______________________________________                                                       WINDING SENSE                                                                 (looking down mandrel from                                     FIBER NUMBERS  right upright toward left upright)                             ______________________________________                                        1-5                CW                                                          6-10              CCW                                                        11                 CW                                                         12-15              CCW                                                        16-20              CW                                                         21-22              CCW                                                        23-25              CW                                                         26-30              CCW                                                        31-33              CW                                                         34-35              CCW                                                        36-40              CW                                                         41-44              CCW                                                        45                 CW                                                         46-50              CCW                                                        51-55              CW                                                         56-60              CW                                                         61-65              CCW                                                        66                 CW                                                         67-70              CCW                                                        71-75              CW                                                         76-77              CCW                                                        78-80              CW                                                         81-85              CCW                                                        86-88              CW                                                         89-90              CCW                                                        91-95              CW                                                          96-100            CCW                                                        ______________________________________                                    

REMOVAL OF FIBER BUNDLE FROM JIG

The following steps should be carried out when removing the fiber bundle14 from the jig 30:

1. Loosely apply thread ties in two locations, both one-half cm. and twocm. from the points of attachment at 1, 2, 3, and 4. Use one colorthread at 1 and 4, and a second color at 2 and 3 to distinguish the twoseparate fiber bundles. The thread tie close to the fiber attachmentsite will later serve to tightly bunch the fibers together; the secondtie is a security measure to group fibers together in the event thatsome fibers escape the primary tie when the fibers are severed from thetape attachment. The ties should be loose slipknots which can later beslipped off the fiber bundle without crimping the fibers.

2. Carefully sever fibers from the points of attachment to tape swatchesusing a razor blade or similar means. Leave no tape clinging to fiberends.

3. Use clean, dry thumb and forefinger to smooth bundle from spreadercollar 33 to cut ends into a compact bundle of parallel fibers. In sodoing, there should be no grossly loose or bowed-out fibers which wouldbe susceptible to breakage when the bundle is later pulled into theshell 11. Tighten the ties nearest the bundle ends after positioning theties as near the ends as possible while still encircling all fibers inthat bundle. Firmly knot these end ties at one end only, i.e., at 1 and2, or 3 and 4. If fibers escape the primary end ties, slide thesecondary loose ties into the appropriate position and tighten.

4. Remove the tape switches from the uprights 31. Remove externalslip-on collars 34 from the mandrel 32. Remove the mandrel-bundleassembly from the jig 30 by sliding the uprights 31 apart. Place themandrel-bundle assembly on a clean, dry working surface.

5. Apply appropriately colored thread leaders 20 cm. in length to thetwo bundle ends which were previously firmly knotted (as per step 3).These leaders should be tightly knotted in the same position as theprimary end ties, and tied such that a long leader extends from one sideof the knot.

6. Trim the fiber bundle ends from step 5 to within two mm. of the endtie. Trim the loose ends of the ties, except the two long leads, asclose as possible to the knots. Remove the loose, secondary ties withforceps, being careful not to damage any fibers. The primary andsecondary ties must remain in position at the other end of the bundle.

7. Use forceps or similar means to carefully remove the nested spreadercollar 33 from the mandrel 32 nearest the bundle end prepared as insteps 5 and 6. Then grasp the two long leads between the fingers of thesame hand and slowly ease the bundle 14 off the mandrel 32, evenlydistributing tension between the two leads. Remount the spreader 33 andexternal collars 34 on the mandrel 32.

INSERTION OF FIBER BUNDLE INTO SHELL

The steps employed in inserting the fiber bundle 14 into the shell 11are as follows:

1. Slip the two Silastic connecting sleeves 13 onto the ends of the CCUshell 11. Slip a dual-circuit endpiece 12 into the connecting sleeve 13at one end of the shell 11 so that the ends of the shell 11 and endpiece12 are held tightly together. With the endpiece 12 lying flat on thesurface, the inoculation ports 16 should point vertically upward.

2. Thread the two long leads from the bundle 14 into the end of theshell 11 without the endpiece. The object is to have one lead projectingfrom one port of the connected endpiece 12 and the second leadprojecting from the second port. The leads should not cross over oneanother within the shell 11.

3. Take up tension on the long leads by gently pulling on the projectingends. The two ends of the bundle 14 should be simultaneously pulled intothe shell 11 through the rubber connecting sleeve 13. It may benecessary to squeeze together the two ends with thumb and forefinger toensure this. Then, very gently draw the bundle 14 into the shell 11,pulling the two leads so that the two ends travel at the same speed,until the ends project out of their respective endpiece ports about onecm. It is imperative that no fibers be crimped and broken during thisprocess.

4. Some rearrangements must be made at the end of the bundle 14 whichhas not yet been fed through an endpiece. It is necessary to incorporateanother one-half twist in this end of the bundle, following the"natural" twist in the body of the bundle so that the two ends of eachof the separate bundled circuits lie on the same side of the CCU shell11. rather than being diagonally opposed as on the fiber bundle jig 30.This is accomplished by positioning fiber bundle ends tied with the samecolor directly opposite one another.

5. To prevent fiber breakage during installation of the second endpiece12 following incorporation of the extra half-twist, it is necessary tore-tie the fiber bundle ends at this end of the bundle 14. Working onone bundle at a time, check the orientation of the ends as per step 4.Remove the primary end tie carefully with forceps. Using clean, drythumb and forefinger, smooth the fiber bundle into a compact form, withno protruding fibers, by gently stroking the bundle from the connectingsleeve 13 toward the bundle end. The loose, secondary tie is slippedgradually toward the bundle end and tightened when the bundle issatisfactorily coherent. Then, a ten cm. lead of appropriate color isknotted onto the bundle end to be used in guiding the bundle 14 into thesecond endpiece 12. The fiber bundle end is then trimmed to within 2 mmof the end tie.

6. Finally thread the two leads into the second endpiece 12 and outseparate ports. Carefully draw the two bundle ends into the commonentrance simultaneously, being careful not to pull the bundle ends atthe other end of the shell 11, back into the shell 11. Gently work theendpiece butt into the connecting sleeve 13, adjusting bundle positionby means of the end leads so that no fibers are crimped and broken.Carefully push the endpiece 12 into the connecting sleeve 13 until thesleeve 13 holds the shell 11 and endpiece 12 tightly together. Adjustbundle position so that roughly equal lengths of fiber bundle protrudefrom the connector at each end. Check that bundle ends of the same colorare directly, rather than diagonally, opposed.

POTTING THE CELL CULTURE UNIT

The steps to be carried out in potting the cell culture unit 10 are asfollows:

1. Make new tight ties (the color of thread is now irrelevant) at allfour bundle ends about 3 mm. from the ends of the endpieces 12. Clip offexcess bundle length about 4 mm away from these ties. Use a solderingiron to melt down the bundle ends back to the ties, thus sealing allfiber lumens for the potting process. Trim loose ends of ties flush.

2. Prepare a well-mixed mixture of a potting composition such as 24 gm.General Electric RTV-11 silicone rubber and 16 gm. Dow-Corning 360Medical Fluid. This amount will pot fifteen headers (four per CCU).

3. Take a 20 cm. length of 1/4"×3/8" O.D. Silastic tubing and carefullyslip one end over each header at one end of the CCU 10. The unit can besuspended at the proper height for potting by passing a string throughthe loop provided by this tubing.

4. Seal off a third header by slipping a 3 cm. length of 1/4" I.D.×3/8"O.D. Silastic tubing over the header and applying a pinch clamp to theopen end of the tubing. To seal the injection port closer to the endsealed with the tubing loop, slip on a 7 cm. length of 1/16" I.D.Silastic tubing with a tightly knotted end. A 65 cm. length of 1/16"I.D. Silastic is slipped over the second inoculation port 16 (the portcloser to the header to be potted), and it is provided with a pinchclamp. Mouth suction should be applied to this tube to draw up pottingcomposition 24 into the open header.

5. The CCU 10 is suspended so that the open header is perpendicular toand about 6 mm from the benchtop. A syringe (without needle) may be usedto draw up and dispense 3 ml of potting mixture 24 into a disposableplastic 3 ml titration cup.

6. One drop of stannous octoate catalyst (General Electric Co.) is addedto the 3 ml. of potting mixture 24, which is then thoroughly mixed withthe tip of a Pasteur pipette (10 sec) and then positioned so that theopening of the header is submerged to one-half the depth of the mixture24. Mouth suction is slowly applied to the suction tubing to draw thepotting material 24 up to the level of the bifurcation of the glassheader. The suction tubing is then clamped off and the mixture isallowed to take its initial set (5-10 min.). This step should beperformed quickly, since one drop of catalyst will rapidly render 3 mlof RTV composition gummy.

7. To pot the remaining three headers, follow a similar procedure, i.e.,place clamped tubing over un-potted headers and suspend the open headerto be potted in a titration cup containing 3 ml of freshly-catalyzed RTVrubber.

In carrying out this procedure, it should be noted that, at each end ofthe CCU:

(a) The RTV in the first header to be potted is drawn up only to thebifurcation of the header, so that the main body of the endpiece doesn'tseal off and prevent potting of the second header; and

(b) The RTV in the second header is drawn up about 5 mm past the end ofthe main polycarbonate shell 11, but caution must be used to avoidapplying too much suction, which may cause clogging of an injection portwith RTV, or "wicking" of RTV up into the active region of the bundle14.

8. After four hours of curing at room temperature, the titration cupsare slit away from the rubber plugs to allow greater air access forcuring the rubber. After 48 hours of curing, the rubber plugs aretrimmed down and finally cut flesh with the end of the header with asingle slash of a sharp razor blade, revealing the open lumens 25 of thefibers.

The dual circuit, woven artificial capillary bundle has beensuccessfully used to culture dense masses of GH₃ rat pituitary tumorcells (MtTW/5) and to quantify mass transport parameters for diffusionand convection of non-metabolite species through the cell mass byapplying a differential pressure between the two circuits of about 300mm Hg. In the culture of cells, the present device may be employed in aconventional manner, with cell suspension medium and cells to becultured being passed into the shell unit 11 through inoculation ports16. General procedures in this regard are outlined in U.S. Pat. No.3,883,393. Also, the specific type of perfusion circuits to be employedwill depend upon the particular use of the device. One such perfusioncircuit which may be employed in both the circuits of the present deviceis that described in U.S. Pat. No. 3,883,393.

The cell culture device of the present invention may be advantageouslyemployed in the simulation of flow or drainage through lymphaticchannels. It is known that the factors which determine the rate andcomposition of lymph flow in humans and other animals include suchfactors as capillary pressure and tissue pressure. Other factors whichare also important include plasma colloid osmotic pressure, tissuecolloid osmotic pressure, total extracellular fluid volume andpermeability of the capillaries. The general physiology of the lymphaticsystem is described, for example, by A.C. Guyton, Textbook of MedicalPhysiology, 2d. Edition, published by W. B. Saunders Co., Phila., Pa.,pp. 52-71, incorporated herein by reference.

Thus, considering the flow system which exists in the body between thecapillaries and the lymphatic channels, with the interstitial spaces ortissues interposed therebetween, an analogy can be seen between thissystem and the cell culture device of the present invention, in whichthere are provided two separate perfusion circuits, one of which mayrepresent the capillaries and the other the lymphatic channels and withthe extracapillary space outside of the fibers representing theinterstitial space. Such factors as fluid composition and pressure maybe varied as desired in the perfusion circuits of the present device inorder to simulate various conditions and flow rates.

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the apparatus and methodsas described herein without departing from the spirit and scope of theinvention or sacrificing its material advantages, the forms hereinbeforedescribed being merely preferred embodiments thereof.

The concept of using different pressures in the two perfusion circuitsto produce convection of the nutrient medium in the extracapillary spaceis intended to include both a difference in hydrostatic pressures in theindividual perfusion circuits and osmotic pressures as would result fromdifferential concentrations of poorly diffusing substances such asalbumin within the perfusate media.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for themaintenance of cells and the formation and maintenance of solid tissuesin vitro which comprises:(a) arranging a plurality of capillaries withina chamber, the capillaries having walls which are permeable to nutrientsrequired for cell growth or maintenance and/or cell products and beingarranged with individual capillaries extending in a specific interwovenrelationship with respect to each other, said capillaries dividing thechamber by the walls of the capillaries into an intracapillary spacewithin the capillaries and an extracapillary space outside thecapillaries, the intracapillary space and the extracapillary spacecommunicating with each other only through the walls of the capillaries;(b) connecting a first portion of said interwoven capillaries to a firstperfusion circuit and a second portion of said interwoven capillaries toa second perfusion circuit; (c) introducing living cells into theextracapillary space so that the cells will settle onto the capillaries;and (d) passing perfusate through the intracapillary space of said firstand section portions of said capillaries.
 2. The method of claim 1,wherein said capillaries are interwoven by a method which includes:(a)arranging a jig having left and right upright members with a mandrelsupported therebetween, said mandrel carrying a pair of slidablyattached spreader collars which are positioned so that one of saidspreader collars is substantially equidistant between the midpoint ofthe length of said mandrel and each of said upright members; (b)attaching a first plurality of fibers from a first position on the rightupright to a first position on the left upright, said respective firstpositions being on diametrically opposite sides of said uprights, saidfirst plurality of fibers having two turns around said mandrel in aclockwise sense as viewed from the right upright along the mandrel tothe left upright; (c) attaching a second plurality of fibers from asecond position on the left upright to a second position on the rightupright, said respective second positions being on diametricallyopposite sides of said uprights, said second plurality of fibers havingtwo turns around said mandrel in a counterclockwise sense; and (d)repeating steps (b) and (c) as necessary to achieve the desired numberof woven fibers.
 3. The method of claim 1 further including the step ofsimultaneously inducing different pressures in said first and secondperfusion circuits whereby, convection of perfusate occurs in saidextracapillary space.
 4. The method of claim 3 wherein a hydrostaticpressure differential is effected between said first and secondperfusion circuits.
 5. The method of claim 3 wherein an osmotic pressuredifferential is effected between said first and second perfusioncircuits.